Description of the female, nymph and larva and mitochondrial genome, and redescription of the male of Ixodes barkeri Barker, 2019 (Acari: Ixodidae), from the short-beaked echidna, Tachyglossus aculeatus, with a consideration of the most suitable subgenus for this tick

Ixodes barkeri, a tick with a distinctive ventrolateral horn-like projection on palpal segment 1, was described in 2019 from two male ticks from the Wet Tropics of Far North Queensland, Australia. However, females lie at the core of the taxonomy and subgenus classification of Ixodes; hence, we sought specimens of female ticks, successfully recovering females, plus nymphs and larvae. Mitochondrial genomes are also desirable additions to the descriptions of species of ticks particularly regarding subgenus systematics. So, we sequenced the mt genomes of I. barkeri Barker, 2019, and the possible relatives of I. barkeri that were available to us (I. australiensis Neumann, 1904, I. fecialis Warburton & Nuttall, 1909, and I. woyliei Ash et al. 2017) with a view to discovering which if any of the subgenera of Ixodes would be most suitable for I. barkeri Barker, 2019. The female, nymph, larva and mitochondrial genome of Ixodes barkeri Barker, 2019, are described for the first time and the male of I. barkeri is redescribed in greater detail than previously. So far, I. barkeri is known only from a monotreme, the short-beaked echidna, Tachyglossus aculeatus (Shaw, 1792), from the highland rainforests of the Wet Tropics of Far North Queensland, Australia. Our phylogeny from entire mitochondrial genomes indicated that I. barkeri and indeed I. woyliei Ash et al., 2017, another tick that was described recently, are best placed in the subgenus Endopalpiger Schulze, 1935.


Background
Ixodes barkeri Barker, 2019, was described from two male ticks collected from the short-beaked echidna Tachyglossus aculeatus (Shaw, 1792). One of these ticks was from rainforest of the Peeramon Scrub, Atherton Tableland, Far North Queensland (FNQ), whereas the other male was from an unknown locality in the vicinity of the Atherton Tableland. Ixodes barkeri is distinctive among Australian ticks, especially for its ventrolateral palpal projection, which is found only in an echidna tick from Papua New Guinea, I. zaglossi Kohls, 1960. Ixodes zaglossi, however, has syncoxae, whereas I. barkeri does not, and the ventrolateral palpal projection in I. barkeri is much bigger [1]. However, this extraordinary species, I. barkeri, could not be placed within a higher taxonomic framework, such as the subgenus classification of Ixodes, largely because of the lack of females. Since [1], we have acquired other specimens of I. barkeri: 6 males, 5 females, 34 nymphs and 2 larvae (Table 1, Fig. 1), allowing us to describe the female, nymph and larvae for the first time and to redescribe the male in greater detail to compare and contrast the morphology of I. barkeri with other species of the Australasian Ixodes clade (sensu [2]). We have also described the mitochondrial genome of I. barkeri, enabling inferences of phylogenetic relationships of this species with others in the genus.
The subgenera of Ixodes are morphologically ambiguous and in need of further refinement and testing with genetic data. In this regard, mitochondrial (mt) genomes have been remarkably instructive about the evolutionary history (phylogeny) of ticks (e.g. [3][4][5][6][7]). Thus, we sequenced the mt genomes of I. barkeri and its possible relatives that were available to us (I. australiensis Neumann, 1904, I. fecialis Warburton & Nuttall, 1909, and I. woyliei Ash et al. 2017) with a view to discovering which if any of the subgenera of Ixodes would be most suitable for I. barkeri Barker, 2019; [8] and [1] were not able to place I. woyliei and I. barkeri in a subgenus, respectively.

Material examined
Only field-collected ticks were available for study. The specimens were from the Barker and Barker Collection at the University of Queensland (Qld), the Queensland Museum (QM), the Australian National Insect Collection (ANIC) and the Field Museum of Natural History, Chicago, Illinois, USA (FM) ( Table 1).

Microscopy methods
Ticks were studied using a stereoscopic microscope (Nikon SMZ800N, Nikon Corp., Tokyo, Japan, and Olympus SZX16, Olympus Corp., Tokyo, Japan), compound microscope (Olympus BX53, Olympus Corp., Tokyo, Japan) and scanning electron microscope (JEOL JSM6610LV, JEOL Ltd., Tokyo, Japan). An ocular micrometre was used to measure ticks. Measurements are in millimetres for the adults, micrometres for the juveniles, and are given as the range followed by the mean and the number of specimens measured (n) in parentheses. Colour digital images were taken with a Canon 6D camera (Canon Corp., Tokyo, Japan).
Adobe Photoshop ® software was used to correct images for broken legs and other damaged parts of the tick and to polish the image.

Sequencing and assembly of mitochondrial genomes
Mitochondrial genomes were sequenced and assembled in two ways. First, the mt genome of I. barkeri Barker, 2019, was sequenced at Novogene Singapore and then assembled at the University of Queensland (UQ) by the protocol of [5]. DNA was prepared by us at the University of the Sunshine Coast and the University of Queensland. We extracted DNA from individual ticks and from various pools (groups of up to 3 ticks) of females, males, nymphs and larvae in an effort get adequate DNA for our experiments from all life-stages i.e. females, males, nymphs and larvae (Below we report that adequate DNA was obtained from a male and a pool of 3 nymphs but not from females nor larvae since the females and larvae had not been preserved well). Ticks were cut in half and then incubated at 56 °C for 62 h with Proteinase K to lyse the cells. The QIAGEN DNeasy Blood and Tissue kit was used to extract genomic DNA. The amount of DNA recovered was measured with Nanodrop and Qbit instruments. Groups of ticks that yielded > 200 nanograms (ng) of DNA were sent to Novogene Singapore for de novo library construction and next-generation Illumina sequencing. Groups of ticks with < 200 ng were combined with DNA from a different organism, usually a bird, to reach the minimum threshold of 200 ng of DNA required by Novogene Singapore. At Novogene Singapore, DNA was sonicated to fragment the DNA, and then fragments were end-polished, A-tailed and ligated with Illumina adaptors. DNA fragments were amplified with PCR, using P5 and P7 oligos, to create genomic libraries, which were purified with AMPure XP system. The Illumina Novaseq 6000 sequencing platform was used to generate two giga-bases of nucleotide sequence data (PE 150). We then constructed de novo contig assemblies of Illumina sequences in Geneious Prime [9] by the default assembler of Geneious Prime. Blast-searches of contigs revealed mt genes of ticks; these gene sequences were then assembled until entire mt genomes had been assembled.
Second, the mt genomes of I. australiensis, I. fecialis and I. woylie were sequenced at the Hokkaido University, Japan, and then assembled at the University of Queensland by the protocol of [5]. DNA was extracted from ticks with the NucleoSpin ® DNA Insect (Macherey-Nagel, Germany). Entire mt genomes were then amplified in two overlapping fragments (long-range and short PCRs). Long-range PCR was used to amplify fragments that comprised about 12-13 kb of the mitogenome with the universal primers: mtG_K23 (5'-TCC TAC ATG ATC TGA GTT YAG ACC G-3') and mtG_K25 (5'-AAA ATT CWT AGG GTC TTC TTG TCC -3') or mtG_K26 (5'-ACG GGC GAT ATG TRC ATA TTT TAG AGC-3'). Short PCRs were then used to amplify 1.5-2.5 kb of mt genomes with genus-specific primers. PrimeSTAR ® GXL DNA Polymerase (Takara-Bio, Shiga, Japan) was used to amplify the long mt PCR products, whereas Tks Gflex ™ DNA Polymerase (Takara-Bio) was used to amplify short mt gene fragments as well as nuclear rRNA genes. PCR conditions for PrimeSTAR ® GXL DNA Polymerase were: 45 cycles of 98 °C for 10 s, 60 °C for 15 s and 68 °C for 10 min. PCR conditions for Tks Gflex ™ DNA Polymerase were: 94 °C for 60 s, 45 cycles of 98 °C for 10 s, 55 °C for 15 s, 68 °C for 60 s and a final extension of 68 °C for 5 min. PCR products were examined on 1.2% agarose gels stained with Gel-Red ™ (Biotium, Hayward, CA). PCR products were purified with a NucleoSpin Gel and PCR Clean Up Kit (Takara-Bio). Illumina sequencing libraries were constructed from the PCR fragments from the long-range and short PCR reactions with the Nextera DNA Library Prep Kit (Illumina, Hayward, CA) and were sequenced with the Illumina MiSeq platform with the MiSeq reagent kit v3 for 600 cycles.

Annotation of mitochondrial genomes
Mitochondrial genomes were annotated with Geneious Prime. Protein-coding genes were identified by searches with BLAST [10] for open reading frames. Regions between protein-coding genes were searched with BLAST [10] to find rRNA genes, tRNA genes and control regions. The tRNA that we expected to find but did not find with BLAST was found with the aid of the tRNAscan-SE Search Server v1.21 [11] and the MITOS Web Server [12]. The nucleotide sequences of tRNA genes were confirmed by studying the putative secondary structure of transcripts, as implemented in Geneious Prime [9].

Phylogenetic methods
Phylogenies were inferred by both maximum likelihood (ML) and Bayesian inference (BI) methods implemented in the RAXML-HPC2 v 8.2.12 [13] and MrBayes v3.2.2 [14], respectively. JmodelTest2 v2.1.6 [15] was used to find the optimal substitution model for the nucleotide dataset. The GTR + G + I model was found to be the best fit for our dataset. In all ML and BI runs (experiments), genes were partitioned. Rapid bootstrapping of 1000 replicates of our data was executed in RAXML-HPC2 v 8.2.12 [13]. There were two simultaneous BI runs: 10 million generations sampled every 1000 MCMC steps. For every BI run, four MCMC chains (three heated and one cold) were executed. The first 25% of steps was discarded as burn-in. Tracer v 1.5 [16] was used to observe the effective sample size (ESS) and convergence of independent runs. Phylogenetic trees were displayed in FigTree v 1.4.4 [17]. Branch support in the phylogenetic trees generated by RAXML-HPC2 v 8.2.12 [12] and MrBayes v 3.2.2 [14] was assessed by the bootstrap values and posterior probability values, respectively. All phylogenies were inferred through the CIPRES Science Gateway v.3.3 [18]. Ixodes pavlovskyi Pomerantzev, 1946, a species from the clade of the "other Ixodes" (sensu [2]) was the out-group.         the arrangement is ARNESF. The arrangement in I. (Exo.) fecialis is the first known arrangement in an Ixodidae tick that is different from ARNSEF. Thus, ARNESF might be a synapomorphy for the subgenus Exopalpiger suture between them, narrower proximally and abruptly widening to broadly rounded apex. Hypostome (Fig. 5C) length 0.36-0.40 (0.38; n = 4), width 0.12-0.16 (0.14; n = 4), ratio 2.46-2.85 (2.68; n = 4); club-shaped, widening to broadly rounded apex with medial indentation; base of hypostome approximately at level of base of palpal segment II; dental formula 4/4 (few rows may be 3/3), basal half of hypostome without denticles, denticles sharply pointed.

Remarks
The males of I. australiensis, I. tasmani, I. victoriensis and I. zaglossi have been described [19,[21][22][23][24]. The male of I. barkeri is easily distinguished from the males of the Endopalpiger species by the absence of the syncoxal areas on all coxae (vs. well-developed syncoxae on coxae I-IV in all those species).
The female of I. barkeri resembles only that of I. woyliei by the absence of syncoxal areas on coxae (vs. females of all other Endopalpiger with well-developed syncoxae). The female of I. barkeri can be differentiated from I. woyliei by the scutum and basis capituli dorsally and ventrally without lateral carinae and other longitudinal ridges (vs. lateral carinae and longitudinal ridges present in I. woyliei), the considerably smaller palpal segment I with a long spur on its posterior margin (vs. greatly enlarged palpal segment I with shorter spur on its posterior margin in I. woyliei), 4/4 dental formula on hypostome (vs. 6/6 in I. woyliei) and the long spur on trochanter I dorsally (vs. indistinct spur in I. woyliei).
The nymph of I. australiensis, I. hydromyidis, I. luxuriosus ( [25] wrote that the nymph of I. luxuriosus had not been described, although there is a brief description of it in [26]), I. steini, I. tasmani, I. victoriensis and I. woyliei have been described [8,19,22,24,26]. Unfortunately, all of these published descriptions and illustrations are too brief for confident comparison. Nonetheless, we note that the nymph of I. barkeri has a scutum without lateral carinae (vs. distinct carinae in I. victoriensis), a scutum and basis capituli dorsally and ventrally without distinct longitudinal ridges (vs. with distinct, sharp ridges in I. woyliei), a distinct cornua (vs. no cornua in I. australiensis, I. hydromyidis, I. tasmani and I. woyliei), mostly 3/3 dental formula on the hypostome (vs. 2/2 dental formula in I. hydromyidis and I. tasmani, 4/4 in I. australiensis), external spurs on coxae I-IV (vs. apparently no spurs on coxae in I. hydromyidis, I. luxuriosus, I. steini and I. tasmani) and tarsi I-IV slightly humped subapically, without a notch (vs. strongly humped tarsi with distinct notch in I. victoriensis).
The larvae of I. hydromyidis, I. tasmani and I. victoriensis have been described [22,24,27,28]. Unfortunately, as with the nymphs, all of these published descriptions and illustrations of larvae are too brief for confident comparison. Nonetheless, we note that the larva of I. barkeri has indistinct cornua on the basis capituli dorsally (vs. distinct cornua in I. victoriensis) and has external spurs on coxae I-III (vs. no spurs on coxae in I. hydromyidis and I. tasmani).
Our diagnoses may be broadened and improved once the nymphs and larvae of the other Australasian species of Endopalpiger are redescribed and illustrated accurately.
The phylogeny from these mt genomes indicates that I. barkeri and I. woyliei are best placed in the subgenus

Discussion
We had hoped to extract sufficient DNA for our experiments from all of the putative life-stages of I. barkeri [1] i.e. females, males, nymphs and larvae. Adequate DNA for our experiments, however, was obtained from only a male and a pool of three nymphs but not from females nor larvae since the females and larvae had not been preserved well enough. The mt genomes sequences of the male (GenBank OM302450) and the pool of three nymphs (OL597991) were > 99.96 % identical; thus, the nymphs were certainly I. barkeri [1]. Although, we do not have mt genome sequences from the females or the larvae the evidence that the females and larva are also attributable to I. barkeri [1] is strong on account of the morphological similarity of the females and larvae to the males and nymphs (above and Figs. 2,3,4,5,6,7,8,9). The subgeneric classification of Ixodes is complex and sometimes ignored, probably because some subgenera are defined ambiguously, making species difficult to place in a subgenus. However, the names of the subgenera are valid, represent hypotheses of relationships and deserve closer attention. Previously, neither [8] nor [1] attempted to place I. woylie and I. barkeri, respectively, in a subgenus. We, however, conclude that I. woylie and I. barkeri are best placed in the subgenus Endopalpiger Schulze, 1935 (Fig. 10). Alas, mt genomes from the other species of Endopalpiger were not available to us: (i) I. victoriensis Nuttall, 1916, and I. hydromyidis Swan, 1931, from Australia; (ii) I. acer Apanaskevich, 2020; I. giluwensis Apanaskevich 2020; I. luxuriosus Schulze, 1935; I. mirzai Apanaskevich, 2020; I. planiscutatus Apanaskevich, 2020; I. steini Schulze, 1935; I. stellae Apanaskevich, 2020; and I. zaglossi Kohls, 1960, from Papua New Guinea.
Paul Schulze was a prolific German taxonomist whose life works were reviewed recently [26]. He described 17 entities that are presently considered as subgenera [30], including Endopalpiger in 1935, with Ixodes luxuriosus Schulze, 1935, as the type species (redescribed by [20]). The subgenus Endopalpiger was based mainly on their prominent and distinctive palps. Later, Schulze [31] gave generic status to Endopalpiger, thus emphasizing the very unusual form of the palps. [32] and [19] considered the subgenus Endopalpiger to be valid, but [33] and [34] presented the subgenus Endopalpiger as a synonym of Exopalpiger Schulze, 1935, but without evidence or argument. Here, our phylogenetic trees show that Endopalpiger and Exopalpiger are not closely related. Rather, Exopalpiger is much closer to Sternalixodes and Ceratixodes than it is to Endopalpiger (Fig. 10).
The four species of Endopalpiger in our tree formed a monophyletic group (barkeri, tasmani, woylie, australiensis); indeed, a monophyletic group with 100% bootstrap support and a posterior probability of 1.0, the highest possible posterior probability (Fig. 10). This is the first phylogenetic tree from entire mt genomes (about 15,000 bps) or any similarly large number of nucleotides. [The only other tree that had more than one species of Endopalpiger was by [8] (Fig. 10; ca. 800 bps of cox1)]. Therefore, we found strong support for Endopalpiger, albeit with a limited set of taxa. The unique nature of palpal segment (article) I is a morphological synapomorphy of Endopalpiger. As described by [19] (p. 13), the female palpal segment I ("I" in Fig. 5C) is greatly enlarged and projects inwardly and forwardly so that it ensheathes each side of the base of the mouthparts, and ventrally palpal segment I is strongly salient ("ss" in Fig. 5C). The only similar palp morphology in adults is that of Exopalpiger, which, in the words of [19] (p. 13), sounds more like that of Endopalpiger than it actually is. According to [19] (p. 13), the female palpal segment 1 of Exopalpiger is also greatly enlarged, being attached at right angles to the transverse axis of the basis, but does not project inwardly or forwardly and it does not ensheathe any part of the base of the mouthparts; ventrally palpal segment I is salient but not as salient as in Endopalpiger.